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Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes

de Wet, T.J.
Winkler, K.R.
Mhlanga, M.
Mizrahi, V.
Warner, D.F.
Abstract
Mycobacterium tuberculosis possesses a large number of genes of unknown or predicted function, undermining fundamental understanding of pathogenicity and drug susceptibility. To address this challenge, we developed a high-throughput functional genomics approach combining inducible CRISPR-interference and image-based analyses of morphological features and sub-cellular chromosomal localizations in the related non-pathogen, M. smegmatis. Applying automated imaging and analysis to 263 essential gene knockdown mutants in an arrayed library, we derive robust, quantitative descriptions of bacillary morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate that functionally related genes cluster by morphotypic similarity and that this information can be used to inform investigations of gene function. Exploiting this observation, we infer the existence of a mycobacterial restriction-modification system, and identify filamentation as a defining mycobacterial response to histidine starvation. Our results support the application of large-scale image-based analyses for mycobacterial functional genomics, simultaneously establishing the utility of this approach for drug mechanism-of-action studies.
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Date
2020-11-06
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Publisher
Elife Sciences
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Keywords
CRISPRi , Mycobacterium tuberculosis , UMAP , Genetics , Functional genomics , Genomics , Infectious disease , SDG-03 Good health and well-being
Citation
de Wet TJ, Winkler KR, Mhlanga M, Mizrahi V, Warner DF. Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes. Elife. 2020 Nov 6;9:e60083. doi: 10.7554/eLife.60083.
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